Volume 14, Issue 7, Pages 824-834 (July 2007) Rational Design, Synthesis, and Biological Evaluation of Progesterone-Modified MRI Contrast Agents  Jiyoun Lee, Joanna E. Burdette, Keith W. MacRenaris, Devkumar Mustafi, Teresa K. Woodruff, Thomas J. Meade  Chemistry & Biology  Volume 14, Issue 7, Pages 824-834 (July 2007) DOI: 10.1016/j.chembiol.2007.06.006 Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 1 Structures of Progesterone-Conjugated MRI Contrast Agents Chemistry & Biology 2007 14, 824-834DOI: (10.1016/j.chembiol.2007.06.006) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 2 Synthesis of Progesterone-Modified Gd(III) Chelate Conjugates (A) Synthesis of neutral conjugates containing no spacer between the steroid and contrast agent. (B) Synthesis of neutral conjugates containing a six-carbon spacer. (C) Synthesis of charged progesterone conjugates. Chemistry & Biology 2007 14, 824-834DOI: (10.1016/j.chembiol.2007.06.006) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 3 Cellular Uptake Studies of Progesterone-Modified Contrast Agents (A) Progesterone Gd(III) chelates are dose dependently absorbed into mammalian breast cancer cells that either express progesterone receptor or are receptor negative. Cells were incubated with 50, 5, 0.5, or 0.05 μM contrast agents for 24 hr, washed with PBS, and lysed, and the amount of intracellular Gd(III) was quantified using ICP-MS. Data represent the average of triplicate determinations divided by the amount of cellular protein to normalize content based on cell number. (B) Progesterone Gd(III) chelates are time dependently absorbed into mammalian breast cancer cells with and without progesterone receptors. Cells were incubated with 50 μM contrast agents for 1, 2, 4, or 24 hr, washed with PBS, and lysed, and the amount of intracellular Gd(III) was quantified using ICP-MS. Data represent the average of triplicate determinations divided by the amount of cellular protein to normalize content based on cell number. (C) Progesterone gadolinium chelates are selectively retained in progesterone receptor-expressing cells at specific time points after leaching into culture medium. Cells were incubated with 50 μM contrast agents for 24 hr, washed with PBS at 0.5, 1, 2, 4, 6, 24, and 48 hr, lysed, and the amount of intracellular and effluxed Gd(III) was quantified using ICP-MS. Data represent the average of triplicate determinations with the amount of Gd(III) in the cell divided by the amount in the media and then divided by the amount of cellular protein to normalize content based on cell number (x axis, incubation time; y axis, retention ratio). Chemistry & Biology 2007 14, 824-834DOI: (10.1016/j.chembiol.2007.06.006) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 4 Progesterone Gd(III) Chelates Function Biologically to Initiate Gene Transcription of a Progesterone Responsive Element T47D cells were transiently transfected with the 3×PRE-luciferase construct and serum starved for 24 hr before treatment with contrast agents for an additional 24 hr. Relative light units were calculated and the data represent the average ± the standard deviation from triplicate measurements. Chemistry & Biology 2007 14, 824-834DOI: (10.1016/j.chembiol.2007.06.006) Copyright © 2007 Elsevier Ltd Terms and Conditions

Figure 5 In Vitro MRI Results (A) T1-weighted images of breast cancer cells incubated with compounds 1 and 2 for 24 hr. Gd(III) chelates demonstrate enhanced cell relaxivity in vitro. The images were obtained at 9.4 T, field of view = 1.5 cm, TE = 10 ms, TR = 200 ms (50 μM), TE = 10.2 ms, TR− 600 ms (500 μM). (B) T1 data of cells incubated with 50, 150, and 500 μM 1 and 2 (T1 data were taken at 9.4 T, 20°C). Chemistry & Biology 2007 14, 824-834DOI: (10.1016/j.chembiol.2007.06.006) Copyright © 2007 Elsevier Ltd Terms and Conditions